sign in sign up
<<
Home , Retigabine, Topiramate

Journal of Pre-Clinical and Clinical Research, 2017, Vol 11, No 1, 61-65 ORIGINAL ARTICLE www.jpccr.eu

Interactions of  with in the mouse tonic-clonic seizure model and chimney test – an isobolographic analysis Mirosław Zagaja1, Barbara Miziak2, Maria W. Kondrat-Wróbel2, Marta Andres-Mach1, Paula Wróblewska-Łuczka1, Piotr Adamczuk1, Robert Chmura3, Stanisław Jerzy Czuczwar2, Jarogniew J. Łuszczki4 1 Isobolographic Analysis Laboratory, Institute of Rural Health, Lublin, Poland 2 Department of Pathophysiology, Medical University, Lublin, Poland 3 Department of Biostatistics, Demography and Epidemiology, Institute of Rural Health, Lublin, Poland 4 Department of Pathophysiology, Medical University, Lublin, Poland Isobolographic Analysis Laboratory, Institute of Rural Health, Lublin, Poland Zagaja M, Miziak B, Kondrat-Wróbel MW, Andres-Mach M, Wróblewska-Łuczka P, Adamczuk P, Chmura R, Czuczwar SJ, Łuszczki JJ. Interactions of retigabine with topiramate in the mouse tonic-clonic seizure model and chimney test – an isobolographic analysis. J Pre-Clin Clin Res. 2017; 11(1): 61–65. doi: 10.26444/jpccr/74557 Abstract Introduction and objectives. Nowadays, one of the treatment options for patients with refractory is polytherapy with two or more antiepileptic drugs (AEDs). Retigabine (RTG) is a novel third-generation AED with unique molecular mechanisms of action that has recently been approved as an add-on drug for the treatment of tonic-clonic seizures. To characterize types of interactions between RTG and topiramate (TPM – a second-generation AED), the maximal electro- shock-induced seizure model (MES) and chimney test in mice were used. Materials and method. In the MES model, the effects of the drugs in terms of suppression of tonic-clonic seizures in male albino Swiss mice were assessed. In the chimney test, the acute neurotoxic effects of the drugs with respect to impairment of motor coordination were determined. Type I isobolographic analysis for the combination of RTG and TPM was applied to assess the anticonvulsant and neurotoxic effects in both the MES and chimney tests. Total brain concentrations of RTG and TPM were measured to exclude any pharmacokinetic interaction between drugs. Results. The type I isobolographic analysis of interaction revealed that the combination of RTG with TPM produced additive interaction in the MES test and additivity, with a slight tendency towards antagonism in terms of acute neurotoxic effects in the chimney test. Neither RTG nor TPM mutually affected total brain concentrations in the experimental animals. Conclusions. The isobolographically analyzed combination of RTG with TPM is favourable and may be recommended to some patients with refractory epilepsy. Key words maximal electro-shock, , isobolographic analysis, Retigabine, Topiramate

INTRODUCTION profiles in both preclinical studies on animals and epileptic patients [2, 4]. The most favourable combinations of AEDs are Nowadays, the efficacious treatment of patients with epilepsy those reported in preclinical studies of synergistic interaction is a challenging issue for clinicians because of the various with respect to seizure suppression and/or antagonistic types of seizures and availability of dozens of antiepileptic interaction with respect to side (neurotoxic) effects 5,[ 6]. drugs (AEDs). 30% of epilepsy patients are still inadequately Retigabine (RTG – a third-generation AED) has been treated with the currently available AEDs [1], and therefore, approved for clinical use as an add-on drug in partial these patients need the therapeutic application of two or onset seizures in adult patients with epilepsy, where other more AEDs [2]. It is obvious that every combination of AED combinations proved to be insufficient to terminate AEDs produces pharmacodynamic, pharmacokinetic or seizures [7]. RTG has a unique molecular mechanisms of both interactions. To terminate seizures in epileptic patients, action owing to properties of a selective M-current potassium clinicians are sometimes obliged to combine various and a subtype selective modulator of GABAA AEDs without any previous information about the type receptors [8, 9]. of interactions between the combined drugs. To provide In previous preclinical studies, the combination of RTG clinicians with necessary information about the perfect with (VPA) exerted supra-additive (synergistic) selection of AEDs in combination, experiments carried out interaction in the mouse maximal electro-shock-induced on animals can properly assess the types of interactions seizure (MES) model [10]. The combinations of RTG with between the AEDs [3]. Overwhelming evidence indicates that (CBZ), (LTG), several two-drug combinations of AEDs displayed favourable (OXC) and (PHT) exerted additive interaction in the mouse MES model [10–12]. The combination of RTG with Address for correspondence: Jarogniew J. Łuszczki, Isobolographic Analysis Laboratory, Institute of Rural Health, Jaczewskiego 2, PL 20-950 Lublin, Poland (LEV) exerted both additive and supra-additive E-mail: [email protected] interactions in the mouse MES model [13]. Additionally, Received: 30.05.2017; accepted: 08.06.2017 the combinations of RTG with CBZ, LEV, LTG and VPA 62 Journal of Pre-Clinical and Clinical Research, 2017, Vol 11, No 1 Mirosław Zagaja, Barbara Miziak, Maria W. Kondrat-Wróbel, Marta Andres-Mach, Paula Wróblewska-Łuczka, Piotr Adamczuk et al. Interactions of retigabine… have recently proved to be clinically effective as the add-on The mice receiving RTG and TPM in high doses displayed therapies in patients with partial-onset seizures [14]. and impairment of motor performance, which were The aim of this study was to perform experiments on quantified with the chimney test 17].[ In this test, the mice animals to characterize the preclinical profile of the had to climb backwards up a transparent plastic tube (3 cm combination of RTG and topiramate (TPM – a second- inner diameter, 30 cm length) within 1 min., as described in generation AED with multiple molecular mechanisms of more detail elsewhere [11, 12]. The acute neurotoxic effects action) in the MES-induced seizure model and chimney of RTG and TPM observed in the animals in the chimney test in mice by the use of type I isobolographic analysis. test were expressed as their median toxic doses (TD50 values), The choice of TPM and RTG for the combination study i.e., doses of AEDs that disturbed motor coordination in was based on the theoretical supposition that their various 50% of the tested animals. The animals received increasing molecular mechanisms of action can be mutually effective. doses of RTG and TPM to obtain an increasing percentage Furthermore, TPM is clinically used in patients with tonic- of impairment of motor coordination that allowed the clonic seizures and partial onset seizures [15]. construction of dose-response effect lines for RTG and TPM. It is widely accepted that the MES model is considered Similarly, the neurotoxic (adverse) effects of the mixture of to be an experimental model of tonic-clonic seizures and RTG with TPM were determined and expressed as the TD50 exp partial onset of in humans [16]. The chimney value, i.e., the dose of the mixture required to impair motor test is considered to be a model assessing acute adverse coordination in 50% of mice subjected to the chimney test. (neurotoxic) effects in experimental animals with respect to This experimental procedure has been described in more the impairment of motor coordination [17]. To exclude any detail elsewhere [11, 12, 19]. pharmacokinetic interactions, total brain concentrations of both, RTG and TPM were determined. Type I isobolographic analysis. Interactions between RTG and TPM in the MES-induced seizure and chimney test were isobolographically analyzed according to the methodology MATERIALS AND METHODS as described elsewhere [10–12, 20]. After determining the

ED50 and TD50 values for RTG and TPM administered alone, Animals. Adult male albino Swiss mice (weighing 22 – median additive doses for the mixture of RTG with TPM (i.e.,

26 g) were used in this study. All experimental procedures ED50 add and TD50 add) were calculated. Subsequently, the test described complied with the ARRIVE guidelines, approved for parallelism of dose-response effects for RTG and TPM was by the local Ethics Committee at the Medical University used independently for both the MES and chimney tests [6, in Lublin, and conformed to the Guide for the Care and 20]. In this study, two parameters were calculated: protective Use of Laboratory Animals (License No.: 28/2007). Each indices (PI) and benefit indices (BI). The PI were ratios of experimental group consisted of 8 mice. TD50 values from the chimney test and the respective ED50 values from the MES model. The PI reflected a satisfactory Drug administration. RTG (GlaxoSmithKline, Brentford, margin of safety between the anticonvulsant and neurotoxic

UK) and TPM ( AG, Schaffhausen, Switzerland) were doses of RTG and TPM [21]. The BI were ratios of PI exp values suspended in a 1% solution of Tween 80 (Sigma-Aldrich, (determined experimentally) and PI add values (theoretically Poznań, Poland) in distilled water and administered predicted to be additive). The BI reflected advantages for intraperitoneally (i.p.) in a volume of 5 ml/kg body weight. the combination of RTG with TPM, considering both RTG was administered 15 min. and TPM 60 min. before all the anticonvulsant and neurotoxic effects exerted by the experimental tests. combination [6, 19]. It is widely accepted that a BI higher than 1.3 indicates a favourable combination that can be Maximal electro-shock seizure (MES) test. Maximal electro- recommended to further clinical practice [6, 19]. convulsions in mice were produced by an alternating current (50 Hz, 25 mA, 500 V, 0.2 s stimulus duration) delivered Measurement of total brain AED concentrations. via ear-clip electrodes, and the tonic hind limb extension Measurement of total brain concentrations of RTG and TPM (seizure activity) in animals was taken as the endpoint. The was undertaken at doses of the drugs corresponding to the anticonvulsant potency of RTG and TPM was determined ED50 exp value for a two-drug mixture at the fixed-ratio of as their median effective doses (ED50 values), i.e., doses of 1:1 from the MES test. Total brain concentrations of RTG AEDs that suppressed 50% of the animals tested against were estimated with high-pressure liquid chromatography maximal electro-convulsions, as described in more detail (HPLC) and those of TPM with fluorescence polarization elsewhere [10–12, 18]. The animals received increasing doses immunoassay (FPIA), as described in more detail elsewhere of RTG and TPM to obtain an increasing percentage of [10–12, 22]. Total brain concentrations of RTG and TPM are protection from tonic hind limb extension that allowed the expressed in μg/ml of brain supernatants. construction of dose-response effect lines for RTG and TPM. The anticonvulsant potency of the mixture of RTG with Statistical analysis. Log-probit analysis was used to calculate

TPM was determined and expressed as the ED50 exp value, ED50, ED50 exp, TD50 and TD50 exp values (with their S.E.M.) for i.e., the dose of the mixture required to suppress tonic hind RTG and TPM administered alone and in combination in limb extension in 50% of the mice. This procedure has been the MES and chimney tests [23]. Subsequently, the unpaired described in more detail elsewhere [11, 12]. Student’s t-test was used to statistically compare the ED50 exp and TD50 exp values with their respective ED50 add and TD50 add Chimney test. Motor coordination impairment in mice values, as reported earlier [24]. Total brain concentrations was evoked by RTG, TPM and their combination, when were statistically analyzed using the unpaired Student’s t-test. the drugs were administered in high (neurotoxic) doses. P<0.05 was considered to be statistically significant. Journal of Pre-Clinical and Clinical Research, 2017, Vol 11, No 1 63 Mirosław Zagaja, Barbara Miziak, Maria W. Kondrat-Wróbel, Marta Andres-Mach, Paula Wróblewska-Łuczka, Piotr Adamczuk et al. Interactions of retigabine…

RESULTS dose-response effects revealed that the mixture of RTG with TPM at the fixed-ratio of 1:1 exerted an additive interaction Anticonvulsant and acute neurotoxic effects of RTG, TPM in both the tonic-clonic seizure model and chimney test in and their combination in the tonic-clonic seizure model and mice (Tab. 1; Fig. 2A-B). The experimentally derived ED50 exp chimney test in mice. Log-probit analysis revealed that RTG and TD50 exp values for the combination of RTG with TPM and TPM, while administered separately, exerted a clear-cut at the fixed-ratio of 1:1 did not significantly differ from the anticonvulsant effects in the MES test in mice (Fig. 1A). The theoretically calculated ED50 add and TD50 add values from both experimentally determined ED50 values from the MES test the lower and upper lines of additivity (Tab. 1; Fig. 2A-B). are presented on a graph (Fig. 1A). Linear regression analysis confirmed that the dose-response lines of RTG and TPM Table 1. Isobolographic analysis of interactions between retigabine (RTG) in the MES test were non-parallel to one another (Fig. 1A). and topiramate (TPM) at the fixed-ratio of 1:1 in the maximal electroshock Similarly, log-probit analysis of RTG and TPM administered (MES)-induced seizure model and chimney test in mice alone revealed that the drugs produced a clear-cut acute ED50 add ED50 exp TD50 add TD50 exp PI exp PI add BI neurotoxic effect in the chimney test in mice (Fig. 1B). The L26.36±9.45 34.94±3.85 L168.6±41.23 509.3±35.40 14.58 6.40 2.27 experimentally derived TD50 values from the chimney test are U45.05±9.09 34.94±3.85 U464.4±59.78 509.3±35.40 14.58 10.31 1.41 present on a graph (Fig. 1B). With linear regression analysis it

Median effective dose (ED50) and median toxic dose (TD50) values (in mg/kg ± S.E.M.) were was found that the dose-response lines of RTG and TPM from calculated by computerized log-probit analysis. The protective indices (PI – as quotients of the chimney test were non-parallel to each other (Fig. 1A). the respective TD50 and ED50 values) for the combination of RTG and TPM were determined experimentally (PI exp) and calculated theoretically (PI add). The benefit indices (BI – as ratios of

PI exp and PI add) determine the application of the combination of RTG and TPM in further clinical practice, considering both, the anticonvulsant and neurotoxic effects.L – indicates ED and Type I isobolographic analysis of interactions between 50 add U TD50 add values from the lower line of additivity; – indicates ED50 add or TD50 add values from the RTG and TPM in the MES model and chimney test in mice. upper line of additivity. The unpaired Student’s t-test was used to statistically analyze the data Type I isobolographic analysis of interaction for non-parallel and no significant differences were reported.

Figure 1 A-B. Log-probit analysis of dose-response effects of retigabine (RTG) and topiramate (TPM) administered alone as well as their combination in the maximal electroshock (MES)-induced seizure model and chimney test in mice. Legend to Figure 1A-B: Doses of RTG, TPM and their combination at the fixed-ratio of 1:1 were transformed into logarithms to the base 10. The protective effects produced by the drugs against tonic-clonic seizures in mice were transformed into probits. Each point indicates a group of 8 mice. Linear regression equations characterizing dose-response effects for particular drugs administered alone and in combination are presented on the graphs. In the MES and chimney tests, the dose-response effect lines for both, RTG and TPM were not parallel to one another. The experimentally determined median effective doses (ED50) and median toxic doses (TD50) for the drugs and their combination are presented on the graphs.

Figure 2 A-B. Isobolograms with additive interactions between retigabine (RTG) and topiramate (TPM) in the maximal electroshock (MES)-induced seizure model and chimney test in mice.

Legend to Figure 2A-B: Median effective doses (ED50 ± S.E.M.) and median toxic doses (TD50 ± S.E.M.) for RTG and TPM are plotted graphically on the X- and Y-axes, respectively. Lower and upper isoboles of additivity represent the curves connecting the ED50 and TD50 values for RTG and TPM administered separately. The points A’ and A” depict the theoretically calculated ED50 add and TD50 add values for both, lower and upper isoboles of additivity. The point M illustrates the experimentally determined ED50 exp and TD50 exp values for total dose of the mixture that produced 50% anticonvulsant effects in the MES test and 50% acute adverse (neurotoxic) effects in the chimney test in mice, respectively. 64 Journal of Pre-Clinical and Clinical Research, 2017, Vol 11, No 1 Mirosław Zagaja, Barbara Miziak, Maria W. Kondrat-Wróbel, Marta Andres-Mach, Paula Wróblewska-Łuczka, Piotr Adamczuk et al. Interactions of retigabine…

It should be mentioned that this study additionally characterized the types of interaction between drugs administered in high toxic doses that evoked acute side- neurotoxic effects, manifesting in form of ataxia and impairment of motor coordination in mice. Results analyzed isobolographically revealed that the combination of RTG with TPM produced additivity with a slight tendency to antagonism in the chimney test – an experimental model, in which the neurotoxic- effects for the AED combination in mice were assessed. Previously, the authors also found that the combinations of RTG with PHT and OXC produced an Figure 3. Effect of topiramate (TPM) on total brain concentrations of retigabine additive interaction with a tendency towards antagonism in (RTG) and inversely, RTG on total brain concentrations of TPM in mice. Legend to Figure 3: Total brain concentrations (means in µg/ml) of RTG and TPM the chimney test [11, 12]. were assayed with high-pressure liquid chromatography (HPLC) and fluorescence A pharmacokinetic study revealed that the combination polarization immunoassay (FPIA), respectively. Doses of RTG and TPM correspond of RTG with TPM, at doses corresponding to the ED50 exp to the ED50 exp value from the MES test (for more details see Table 1). The unpaired Student’s t-test was used to statistically analyze the data and no significant value, was devoid of any significant changes in total brain differences were reported. concentrations of both drugs, since it was found that neither RTG significantly affected total brain concentrations of TPM, Effects of RTG on total brain concentrations of TPM and nor TPM considerably affected total brain concentrations inversely, TPM on total brain concentrations of RTG. of RTG in mice. There is no doubt that this bidirectional Estimation of total brain concentrations of RTG with HPLC measurement of total brain concentrations of both drugs revealed that the co-administration of RTG with TPM was provided the authors with detailed information about associated with no significant changes in total brain RTG pharmacokinetic properties of drugs combined together. Lack concentrations in mice (Fig. 3). Moreover, with FPIA it of any pharmacokinetic interactions between RTG and TPM is was found that total brain concentrations of TPM did not consistent with previously published results. More specifically, significantly differ after concurrent administration of RTG the authors have confirmed the lack of any pharmacokinetic (Fig. 3). interactions between RTG and OXC, PHT, LTG, CBZ and VPA [10–12]. Thus, when translating the results from the current Isobolographic parameters for combinations of RTG study to clinical settings, no pharmacokinetic interactions with TPM. The PI for RTG and TPM administered alone between RTG and TPM are expected in humans. were 1.19 and 15.25, respectively (results not shown). The On the other hand, the molecular mechanisms of action isobolographically calculated BI for the combination of RTG of both AEDs, i.e., RTG and TPM, contributing to the with TPM at the fixed ratio of 1:1 ranged from 1.41–2.27 observed additive interaction in the mouse MES model (Tab. 1). should be discussed briefly. As regards RTG, the drug is a positive modulator of M-current of potassium channels

and, additionally, it potentiates GABAA receptor-mediated DISCUSSION responses [26]. In the case of TPM, molecular studies revealed that the drug blocks voltage-dependent sodium channels,

Results presented in this study indicate clearly that the modulates AMPA/kainate receptors, potentiates GABAA combination of RTG with TPM produced additive interaction receptor-mediated responses, inhibits R-type, but activates in both, the MES model and chimney test in mice. These L-type calcium channels, inhibits II findings are similar to those published earlier for the and IV isoenzymes and changes intra- and/or extracellular combinations of RTG with OXC, LTG, CBZ and PHT at pH [27]. Accumulating experimental evidence indicates the fixed-ratio of 1:1, for which additive interactions were that the combinations of AEDs with diverse and different reported in the mouse MES model [10–12]. In contrast, the mechanisms of action are favourable and result in synergistic combination of RTG with LEV and VPA produced supra- interactions [5]. Unfortunately, despite various and additive (synergistic) interaction in the mouse MES model complementary molecular mechanisms of the anticonvulsant [10, 13]. However, in the case of the combination of RTG activities of RTG and TPM, neither drug synergistically with LEV, the type II isobolographic analysis was used to interacted together. Instead of the expected synergy, the characterize the synergistic interaction between AEDs in additive interaction was observed in the mouse MES model. the mouse MES model [13]. Generally, type II isobolographic It should be stressed that this study evaluated both the analysis is used in preclinical studies on animals if one anticonvulsant and acute neurotoxic (adverse) effects of the tested drugs in mixture does not have any activity produced by the combination of RTG with TPM at the in the studied experimental seizure model. In the case of fixed-ratio of 1:1. Owing to the isobolographic analysis, the the combination of RTG with LEV, the latter drug did not BI could be calculated for the combination of RTG with produce anticonvulsant activity in the mouse MES model; TPM. Generally, the BI allows researchers to classify the therefore, it is considered to be virtually inactive in the interactions with respect to their potential application in mouse MES model [25]. This is the reason for testing the further clinical settings [6, 28]. As recommended earlier, a BI combination of RTG and LEV with type II isobolographic higher than 1.3 indicate clinically favourable combinations analysis. In contrast, with type I isobolographic analysis for of AEDs. Since the BI for the combination of RTG with parallel dose-response lines, the combination of RTG with TPM ranged from 1.41 – 2.27, it can be ascertained that the VPA produced supra-additive (synergistic) interaction in the beneficial two-drug combination is worth recommending mouse MES model [10]. for patients with pharmacoresistant epilepsy. Journal of Pre-Clinical and Clinical Research, 2017, Vol 11, No 1 65 Mirosław Zagaja, Barbara Miziak, Maria W. Kondrat-Wróbel, Marta Andres-Mach, Paula Wróblewska-Łuczka, Piotr Adamczuk et al. Interactions of retigabine…

CONCLUSIONS the chimney test and the model of generalized tonic-clonic seizures in mice. J Epileptol. 2016; 24: 87–94. 12. Żółkowska D, Zagaja M, Miziak B, Kondrat-Wróbel MW, Załuska K, If the results from this study could be translated into clinical Florek-Łuszczki M, et al. Isobolographic assessment of interactions trials, the combination of RTG with TPM would be beneficial between retigabine and phenytoin in the mouse maximal electroshock- for epilepsy patients remaining refractory to currently induced seizure model and chimney test. Health Prob Civil. 2016; 10: available AEDs. 54–59. 13. Luszczki JJ, Zagaja M, Miziak B, Florek-Luszczki M, Czuczwar SJ. Synergistic interaction of retigabine with levetiracetam in the mouse Acknowledgements maximal electroshock-induced seizure model: a type II isobolographic The generous gift of retigabine from GlaxoSmithKline analysis. Pharmacology. 2015; 96: 11–15. (Brentford, UK) is gratefully acknowledged. The authors also 14. Lerche H, Daniluk J, Lotay N, DeRossett S, Edwards S, Brandt C. Efficacy express their thanks to Dr. G. Raszewski (Institute of Rural and safety of ezogabine/retigabine as adjunctive therapy to specified single antiepileptic medications in an open-label study of adults with Health, Lublin, Poland) for the skillful determination of the partial-onset seizures. Seizure. 2015; 30: 93–100. brain concentrations of retigabine. This study was supported 15. NICE guidelines. : diagnosis and management. https://www. by an unrestricted research grant from GlaxoSmithKline nice.org.uk/guidance/cg137 (access: 21.02.2017). (Brentford, UK). 16. Loscher W, Fassbender CP, Nolting B. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. II. Maximal electroshock seizure models. Epilepsy Res. 1991; 8: 79–94. REFERENCES 17. Boissier JR, Tardy J, Diverres JC. Une nouvelle méthode simple pour explorer l’action «tranquillisante»: le test de la cheminée. Pharmacology. 1. Kwan P, Brodie MJ. Early identification of refractory epilepsy. N Eng J 1960; 3: 81–84. Med. 2000; 342: 314–319. 18. Kondrat-Wróbel MW, Łuszczki JJ. Interaction of three-drug combination 2. Stephen LJ, Brodie MJ. Antiepileptic drug monotherapy versus of , carbamazepine and in the mouse maximal polytherapy: pursuing seizure freedom and tolerability in adults. Curr electroshock-induced seizure model – an isobolographic analysis. Opin Neurol. 2012; 25: 164–172. Health Prob Civil. 2016; 10: 55–61. 3. Loscher W. Single versus combinatorial therapies in : 19. Luszczki JJ, Czuczwar SJ. Interaction between lamotrigine and Novel data from preclinical models. Epilepsy Behav. 2015; 49: 20–25. in the maximal electroshock-induced seizures in mice: an 4. Brodie MJ, Sills GJ. Combining antiepileptic drugs--rational isobolographic analysis. Eur Neuropsychopharmacol. 2005; 15: 133–142. polytherapy? Seizure. 2011; 20: 369–375. 20. Luszczki JJ. Isobolographic analysis of interaction between drugs with 5. Deckers CL, Czuczwar SJ, Hekster YA, Keyser A, Kubova H, Meinardi H, nonparallel dose-response relationship curves: a practical application. et al. Selection of antiepileptic drug polytherapy based on mechanisms Naunyn Schmiedebergs Arch Pharmacol. 2007; 375: 105–114. of action: the evidence reviewed. Epilepsia. 2000; 41: 1364–1374. 21. Loscher W, Nolting B. The role of technical, biological and 6. Luszczki JJ, Czuczwar SJ. Preclinical profile of combinations of some pharmacological factors in the laboratory evaluation of anticonvulsant second-generation antiepileptic drugs: an isobolographic analysis. drugs. IV. Protective indices. Epilepsy Res. 1991; 9: 1–10. Epilepsia. 2004; 45: 895–907. 22. Luszczki JJ, Wlaz A, Karwan S, Florek-Luszczki M, Czuczwar SJ. 7. European Medicines Agency. Trobalt: EPAR – Product Effects of WIN 55,212–2 mesylate on the anticonvulsant action of Information. http://www.ema.europa.eu/ema/index.jsp?curl=pages/ lamotrigine, oxcarbazepine, and topiramate against medicines/human/medicines/001245/human_med_001431. maximal electroshock-induced seizures in mice. Eur J Pharmacol. jsp&mid=WC0b01ac058001d124 (access: 12.02.2017). 2013; 720: 247–254. 8. Rundfeldt C, Netzer R. Investigations into the of 23. Litchfield JT, Jr., Wilcoxon F. A simplified method of evaluating dose- the new anticonvulsant retigabine. Interaction with GABAergic and effect experiments. J Pharmacol Exp Ther. 1949; 96: 99–113. neurotransmission and with voltage gated ion channels. 24. Tallarida RJ. Revisiting the isobole and related quantitative methods Arzneimittelforschung. 2000; 50: 1063–1070. for assessing drug synergism. J Pharmacol Exp Ther. 2012; 342: 2–8. 9. Treven M, Koenig X, Assadpour E, Gantumur E, Meyer C, Hilber K, 25. Loscher W. The search for new screening models of pharmacoresistant et al. The anticonvulsant retigabine is a subtype selective modulator of epilepsy: is induction of acute seizures in epileptic rodents a suitable GABAA receptors. Epilepsia. 2015; 56: 647–657. approach? Neurochem Res. 2016; (In press). 10. Luszczki JJ, Wu JZ, Raszewski G, Czuczwar SJ. Isobolographic 26. Rogawski MA, Bazil CW. New molecular targets for antiepileptic drugs: characterization of interactions of retigabine with carbamazepine, alpha(2)delta, SV2A, and K(v)7/KCNQ/M potassium channels. Curr lamotrigine, and valproate in the mouse maximal electroshock-induced Neurol Neurosci Rep. 2008; 8: 345–352. seizure model. Naunyn Schmiedebergs Arch Pharmacol. 2009; 379: 27. White HS, Smith MD, Wilcox KS. Mechanisms of action of antiepileptic 163–179. drugs. Int Rev Neurobiol. 2007; 81: 85–110. 11. Zagaja M, Miziak B, Załuska K, Marzęda P, Drop B, Załuska-Patel K, 28. Luszczki JJ, Czuczwar M, Kis J, Krysa J, Pasztelan I, Swiader M, et al. et al. Additive interactions between retigabine and oxcarbazepine in Interactions of lamotrigine with topiramate and first-generation antiepileptic drugs in the maximal electroshock test in mice: an isobolographic analysis. Epilepsia. 2003; 44: 1003–1013.